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Nascent Polypeptide Chains Exit the Ribosome in the Same Relative In RAPID COMMUNICATIONS Nascent Polypeptide Chains Exit the Ribosome in the Same Relative Position in Both Eucaryotes and Procaryotes CARMELO BERNABEU,*[I ELAINE M. TOBIN,*II AUDREE FOWLER,*{} IRVING ZABIN,*§ and JAMES A. LAKE*l[ * Molecular Biology Institute and II Department of Biology, University of California, Los Angeles, California 90024; and § Department of Biological Chemistry, University of California Medical School, Los Angeles, California 90024. Dr. Bernabeu's present address is Sidney Father Cancer Institute, Boston Massachusetts 02115. ABSTRACT We located the polypeptide nascent chain as it leaves cytoplasmic ribosomes from the plant Lemna gibba by immune electron microscopy using antibodies against the small subunit of the enzyme ribulose-l,5-bisphosphate carboxylase. Similar studies with Escherichia coil ribosomes, using antibodies directed against the enzyme //-galactosidase, show that the polypeptide nascent chain emerges in the same relative position in plants and bacteria. The eucaryotic ribosomal exit site is on the large subunit, ~75 ,g, from the interface between subunits and nearly 160 ,~ from the central protuberance, the presumed site for peptidyl transfer. This is the first functional site on both the eucaryotic and procaryotic ribosomes to be determined. It has been appreciated for some time that, in general, eucar- sites on the large subunits of both ribosomal types. This site is yotic and procaryotic ribosomes share common structural fea- 150 A from the presumed site of the peptidyl-transferase (11). tures (for reviews, see references 1, 2). With the advent of In the duckweed, Lemna gibba, the polypeptide nascent chain immune electron microscopy (3, 4), however, the Escherichia emerges from the large subunit at a region on the side of the coli ribosome has become better understood than its eucaryotic ribosome opposite the translational domain and in the same counterparts. In E. coli, the functional sites involved in trans- relative position as found in the E. coil ribosome. Hence, in lation are clustered into part of the ribosome, comprising spite of the greater complexity of eucaryotic as compared to approximately two-thirds of its volume, referred to as the procaryotic ribosomes, the overall organization of the exit "translational domain." Functional sites contained in the trans- domain on ribosomes, as reflected by the location mapped for lational domain include the initiation factor binding sites (5, the exit site, seems to be similar in both. 6), the messenger binding site (7-9), the peptidyl transferase (10-12), the 5S RNA (13), and the L7/L12 proteins (14) that MATERIALS AND METHODS mediate the GTP-dependent steps of translation (for a review Preparation of Polysomes from E. coil: 2.5 x 10it cellsfrom E. of these locations, see reference 15). Together, these sites define cell A324-5 (17) were resuspended in 2 ml of buffer A (150 mM NH~C1/20 mM the translational domain. Corresponding structural regions are Tris.HCl, pH 7.6/10 raM MgCh) and disrupted in a French pressure ceU at found in eucaryotic ribosomes, as well as in archaebacterial 13,800 psi. The ceU extract was centrifuged in a SS-34 rotor for 8 nun at 8,000 rpm. The supernatant containing the polysomes was layered (0.6 ml per tube) on ribosomes (16). top of a 15-30% sucrose gradient with 0.5 ml of 60% sucrose cushion on the Other aspects of ribosomal organization, particularly those bottom in buffer B (150 mM NFLC1/20 mM Tris.HCl, pH 7.6/5 mM MgCh). involved with protein secretion and processing, could possibly The polysomes were pelleted at 45,000 rpm for 125 min in a SW50.1 rotor differ extensively in eucaryotic and procaryotic ribosomes since (Beckman Instruments, Inc., Spinco Div., Palo Alto, CA). The superuatant was discarded and the pellet rinsed immediately with 5 ml of cold buffer B in order the rough endoplasmic reticulum has no obvious counterpart to eliminate the remaining sucrose. The polysomes were resuspended in buffer B in the procaryotic cell. Here we report investigations on the (400/~1) and stored at -80°C. The yield of polysomes was A~o units per 1011 location of the polypeptide nascent chain as it exits from the cells. ribosome in both procaryotes and eucaryotes. We have mapped Preparation of Polysomes from L. gibba: L. gibba plantsG-3 the exit site of the nascent chain on ribosomes synthesizing the (18) in growth medium were washed with distilled water at room temperature enzymes fl-galactosidase and the small subunit of ribulose-1,5- and poured onto liquid nitrogen or crushed ice at -20°C. -35 g of/.,, gibba were macerated in a mortar and the paste resuspended in 70 ml of extraction buffer bisphosphate carboxylase (Rubisco) using antibodies directed (17% sucrose/0.4 M KC1/30 mM MgCIz/50 mM Tris, pH 9.0). AU the operations against these proteins. were carried out at 4°C. The mixture was ground in a Waling Blender (Waling The exit sites are at a single region located at comparable Productioas Div., Dynamics Corp. of America, New Hartford, CT) four times THE JOURNAL Of C£LL 81OtOGV - VOI.UM[ 96 MAY 1983 1471-1474 © The Rockefeller University Press - 0021-9525/83/05/1471/04 $1.00 1471 for 15 s, with 1-min intervals. The homogenate was poured through two layers and then eight layers of cold miracloth. The solution was spun for 7 rain at 3,000 A B rpm in a SS-34 rotor. The volume of the supematant was recorded and 0A vol of 70S 20% Triton X-100 was added. The mixture was spun for 20 rain at 12,000 rpm in a SS-34 rotor. The supematant was layered, with a wide pipette, on top of 5 ml of sucrose cushion (60% sucrose/0.2 M KC1/20 raM Tris[pH 7.6]/5 mM MgC12), and more extraction buffer was added to equilibrate all the tubes. After centrif- ugation for 3 h at 49,000 rpm in a 50.2 Ti rotor (Beckman Instruments, Inc.), the supernatant was discarded and the pellet washed with resuspension buffer (50 mM KCI/20 mM Tris[pH 7.6]/5 mM MgC12). The pellet was finally resuspended in 200/.d of resuspension buffer. These polysomes can be further purified by centrifugation in a SW 50.1 rotor on a sucrose gradient (0.5 ml of 60% sucrose TOP BOTTOM TOP BOTTOM cushion at the bottom and 15-30% sucrose gradient in buffer C [@2 M KCt, 20 mM Tris and 5 mM MgC12]) by spinning at 45,000 rpm for 2 h. FIGURE 1 Analysisof polysomes. (A) L.gibba. (B) E. coil Polysomes (2.1 A2eo U) were layered onto a 15-30% sucrose gradient in buffer Purification of tbe Proteins: The enzyme ribulose-1,5-bisphosphate carboxylase was purified from L. gibba G-3 (18). 20 g of L. gibba were mixed B ( £. gibba) or A ( E. coil) and centrifuged (5W50.1 rotor) for 30 rain. with 10 ml of buffer D (100 mM KC1, 20 mM Tris-HCl, pH 7.4, and 5 mM at 245,000 g. MgCI2), 6 g ofDowex 1 (I × 200--400) and 20 g of sand. The mixture was ground in a mortar and the suspension of the homogenized plant was fdtered through two and then eight muslin layers. The fdtrate was centrifuged for 20 rain at 10,000 rpm in a SS-34 rotor. The clear superuatant was then centrifuged for 30 70S B rain at 45,000 rpm in a SWS0.1 rotor. The supematant was concentrated by either polyethylene glycol (PEG) in a dialys/s bag or by nitrafdtration in an Amicon apparatus (Amicon Corp., Scientific Sys. Die., Danvers, MA). When the volume was ~5-8 ml, the solution was dialyzed overnight against 100 mM KCI, 20 mM Tris-HCl, pH 7.4, and 5 mM MgCI2. The dialysate (1-2 ml Per tube) was layered on top of a 5-30% sucrose gradient in 25 mM Tris-HC1, pH 7.4, and 15 mM MgClz and centrifuged in a VTi 50 rotor (Beckman Instruments, Inc.) for 160 rain at 49,000 rpm. Under these conditions the ribulose-1,5-bisphosphate carbox- ylase enzyme complex (Mr ~500,000) moves into the gradient ahead of the bulk of other proteins. The Peaks of the enzyme were pooled and dialyzed for 48 h TOP BOTTOM TOP BOTTOM against 2 L of 5% acetic acid (with one change). The acidic solution was then lyophilized. The subunits of the enzyme were separated by preparative SDS FIGURE 2 Isolation of dimers linked by IgG's (A) L. gibba. (B) E. electsophoresis of the lyophilized protein. The small (13,000) and large (52,000) co#. 4 A2eo U of polysomes were reacted with IgG and digested with subunits were localized on the gel by cutting and staining with Coomassie Blue RNase A as described in Materials and Methods. The final mixture a l-cm strip on one side of the slab gel. The unstained gel strips of large and was layered on top of a 15-30% sucrose gradient in buffer 13 (l. small subunits were soaked in water for 3 h in order to remove the SDS. The gels gibba) or A (E. coil) and centrifuged (VTi 65 rotor) for 35 min at were then lyophillzed for 48 h and ground to a free powder in a mortar. The 113,000 g. The shaded dimer peak was collected and negatively enzyme fl-galactosidase was purified as previously described (17). stained With 1% uranyl acetate. Preparation of A n tibodies: To prepare antibodies against the small and large subunits of ribulose-l,5-bisphosphate carboxylase, 1 ml of 0.15 M NaC1, and 0.1 M phosphate buffer, pH 7.0, was added to a gel powder of each previously documented (18, 19).
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